Efficient repair of DNA double strand breaks is essential for the maintenance of chromosomal integrity and persistence of higher organisms. In higher eukaryotes, non-homologous DNA end joining (NHEJ) is the primary pathway that repairs these breaks. NHEJ also functions in developing lymphocytes to repair strand breaks that occur during VDJ recombination, the site-specific recombination process that provides for assembly of functional antigen receptor genes. If VDJ recombination is impaired, B and T lymphocyte development is blocked resulting in the disease, severe combined immunodeficiency [SCID]. In the last decade, an intensive research effort has focused on NHEJ resulting in a reasonable understanding of how DSBs are resolved. Six distinct gene products have been identified which function in this pathway [Ku70, Ku86, XRCC4, DNA ligase IV, Artemis, and DNA-PKcs]. Three of these comprise one complex, the DNA dependent protein kinase (DNA-PK). This protein complex is central during non-homologous end joining because DNA-PK initially recognizes and binds to damaged DNA and then targets other repair activities to the site of DNA damage. Though recent data demonstrate unequivocally that DNA-PK's kinase activity is essential during NHEJ, it is not clear why. That DNA-PK alters the function of one or more important mediator(s) of NHEJ is the central hypothesis to be tested in the proposed studies. Five of the six factors known to function in NHEJ are targets of DNA-PK's kinase activity (either in vitro or in vivo); only DNA ligase IV is not. However, to date there is no compelling evidence that phosphorylation of any of these factors is functionally relevant. The proposed research will map DNA-PK's target phosphorylation sites in each of these five factors. Using a mutational approach, we will then determine whether DNA-PK phosphorylation of each factor is functionally relevant. In sum, since the non-homologous end-joining pathway is critical for the maintenance of genomic integrity, understanding how this pathway functions is of unquestionable relevance. However, our understanding of how nonhomologous DNA end joining works will be incomplete without a clear understanding of what factor(s) are activated by DNA-PK's kinase activity.